Background: The Repeat Expansion Diseases are caused by increases in the number of repeat units in a specific tandem repeat in a single gene. The Fragile X-related disorders (FXDs) arise from expansion of a CGG.CCG-repeat in the 5' UTR of the X-linked FMR1 gene. Carriers of alleles with 55-200 repeats, so-called premutation (PM) alleles, are at risk for a neurodegenerative disorder, Fragile X-associated tremor-ataxia syndrome (FXTAS), and a form of ovarian dysfunction known as FX-associated primary ovarian insufficiency (FXPOI). Furthermore, in females, the PM allele can undergo expansion on intergenerational transfer that can result in their children having alleles with >200 repeats. This expanded allele is known as a full mutation (FM) and, with very few exceptions, all individuals who inherit such alleles have Fragile X syndrome (FXS), the leading heritable cause of intellectual disability and autism. FXS symptoms arise because repeat expansion leads to gene silencing and the subsequent absence of FMRP, the FMR1 gene product, a protein important in many pathways including insulin signaling. The mechanism by which is expansion occurs is thought to differ from the generalized microsatellite instability (MSI) seen in many different cancers in that the instability is confined to a single genetic locus, it shows a strong expansion bias and our work has now shown that genes that normally protect against MSI are actually required to generate the FX mutation. Expanded alleles are also associated with a folate-sensitive fragile site that is coincident with the repeat on the X chromosome. There is reason to think that this fragile site is responsible for the high frequency loss of the affected chromosome resulting in Turner syndrome (45, X0) in female carriers of a FM allele. Progress report: Our previous work using a FX mouse model we developed (Entezam et. al., 2007), has shown that Polbeta together with the mismatch repair complexes, MutSbeta and MutSalpha are actually responsible for the mutation that results in the FXDs (Lokanga et. al., 2012; 2015; Zhao et. al., 2014; 2015; 2016; Zhao and Usdin, 2015). In this reporting period, we have extended our studies to examine other proteins in an attempt to better understand how normal DNA repair processes become subverted to produce these mutations. Amongst our findings in this reporting period is the discovery that MutLgamma, a protein with a minor role in mismatch repair but a major role in resolving Holliday Junctions during meiosis, is also required for expansions (Zhao et. al., 2018). In contrast, EXO1 and FAN1, 2 5'-3' exonucleases protect against expansion (Zhao et. al., 2018; Zhao and Usdin, 2018a). In particular, we showed that EXO1 protects against expansion in 2 different ways, one that depends on its nuclease activity and one that does not. That has intriguing implications for the expansion mechanism. A role for FAN1 is interesting since polymorphisms in the human FAN1 gene are amongst the strongest modifiers of the age at onset and disease severity in a number of other Repeat Expansion Diseases. This finding supports that our contention that our findings in mice are relevant for our understanding of expansions in humans as well and adds to the growing body of evidence suggesting that the different Repeat Expansion Diseases share a common mutational mechanism. We also showed that LIG4, a DNA ligase essential for Non-Homologous End-Joining (NHEJ), a form of double-strand break repair, also protects against expansion (Gazy et. al, 2018). This provides the first evidence for a double-strand break (DSB) intermediate in the expansion process and thus for the role of a form of DSB repair other than NHEJ in generating expansions. We also demonstrated that in mice expansions are much less extensive in blood than in brain and gonads. This may have implications for humans where blood is often the source of DNA for diagnosis. In addition, not only does the expansion rate vary between different organs, but even within the same organ, some cell types expand and other do not. For example, expansion in liver is limited to hepatocytes (Gazy et. al, 2018). In male gonads, expansion is confined to the replicating spermatogonial stem cells, while in females, expansion occurs in non-dividing oocytes (Zhao and Usdin, 2018b). Given that CGG-repeats are very difficult to replicate (Usdin and Woodford, 1995; Yudkin et. al., 2014), expansion in replicating vs non-replicating germ cells in males and females could explain why FM alleles only transmitted maternally and why FM males only have PM alleles in sperm. Expansion in non-dividing oocytes also supports a model for expansion that does not involve aberrant chromosomal replication. In this review period we have also built on our previous experience with the development of assays for FXS (Hayward and Usdin, 2017), to develop a diagnostic test for a new Repeat Expansion Disease, GLS Deficiency (van Kuilenberg et. al., 2019). This disorder results from a large CAG-repeat expansion in the GLS gene and results in a deficiency of glutaminase, the enzyme required for the conversion of glutamine to glutamate, an essential neurotransmitter. As with FXS, amplification of long CAG-repeats is challenging. However, we showed that our assay is able to reliably amplify even alleles with >1000 repeats.